GSM5750827: briggsae-lcap-glp1-ya-rep2; Caenorhabditis briggsae; RNA-Seq
RNA was extracted from one million pelleted nuclei by adding 500ul Trizol and 100 µl of Chloroform and shaking samples vigorously for 15 s. Samples were then spun at 12,000 g for 15 min at 4 C and the aqueous phase was transferred into one volume of ice-cold Isopropanol. 0.5 µl of GlycoBlue (AM9515) was added and RNA was left to precipitate overnight at -20 C. Once precipitated, RNA was pelleted (12,000 g for 30 min at 4 C), washed in 1 ml of ice-cold 80% Ethanol and resuspended in 12 µl of RNAse-free water. Concentration was estimated using Qubit. A minimum of 20 ng of total nuclear RNA was used to make long nuclear RNA-seq libraries. Long nuclear RNA (>200 nt) was isolated using Zymo Clean and Concentrate columns (#R1013) and stranded libraries were prepared using the NEB Next Ultra Directional RNA Library Prep Kit (#E7420S) after removing rRNA using the Ribo-Zero rRNA removal kit (MRZH11124). Long nuclear RNA-seq libraries were made from two biological replicates for each tissue.